The establishment and maintenance of epithelial cell polarity is central to the normal function of epithelial organs; the ability of the intestinal epithelium to serve as a selective barrier to antigens and pathogens while absorbing nutrients relies on the presence of intact cell junctions and distinct apical and basolateral membrane domains. Also, loss of polarity is an important hallmark of many epithelial cancers and has been correlated with transformation and uncontrolled growth. Our investigations of intestinal development and epithelial polarity have focused on the integral membrane protein, endotubin. Endotubin is found in apical endosomes of polarized epithelial cells. It is expressed at high levels in developing epithelial organs such as intestine, liver, kidney and lung, implying a critical role in the development of epithelial structures. Moreover, we have evidence from in vitro studies that endotubin regulates junctional integrity and epithelial polarity. In the previous funding period, we discovered that the small GTPase Rab14 interacts with the cytoplasmic domain of endotubin and that mutant forms of Rab14 compromise targeting to the apical domain. In this proposal, we describe experiments to elucidate the mechanism of action of endotubin and Rab14 in the establishment of epithelial cell polarity during development and in the loss of polarity during transformation. Our hypothesis is that endotubin and Rab14 regulate the establishment and maintenance of polarity in epithelial cells and that loss of function of these proteins results in loss of barrier function and/or apical-basolateral polarity, leading to compromised immunity in the neonate, increased susceptibility to inflammatory bowel disease, and cancer. Relevance to human disease: Maintenance of epithelial cell polarity is essential for intestinal epithelial barrier function, and loss of polarity may predispose these cells to transformation and uncontrolled growth. Understanding how epithelial cells generate and maintain their polarized phenotype is critical for our understanding of intestinal development and pathology.